Francisella tularensis is considered a Category A bioweapon due to the ease of transmission, the low infectious dose and high mortality associated with pneumonic tularemia, and the fact that it has been intensively studied and developed in bioweapons programs in several countries. There is currently no vaccine approved for general human use. A live vaccine strain (LVS) developed from F. tularensis subsp. holarctica has been shown to be very efficacious, however, the mechanism of attenuation in LVS is unknown, making it unlikely to be approved for general use. The focus of this research effort is to identify potential live vaccine candidates of F. tularensis subsp, holarctica that can protect against aerosol F. tularensis subsp, tularensis, and then thoroughly characterize their pathogenic potential and the immune response to them. This will dramatically increase our understanding of the virulence of F. tularensis and the immune response to it, and also identify live vaccine candidates and illuminate what constitutes protective immunity to tularemia. First, every gene of F. tularensis subsp. holarctica will be mutated and recombined back onto the chromosome. The viable mutant bacteria will be combined in pools and inoculated subcutaneously into mice, and those mutants that cannot survive within the host will be identified by a microarray-based technique. Next, the attenuated F. tularensis subsp, holarctica mutants will be used to vaccinate mice, which will then be challenged with aerosolized F. tularensis subsp, tularensis; strains that protect against subsp. tularensis aerosol challenge are potential live vaccine candidates. These strains will be tested for markers of virulence: ability for intramacrophage survival and growth, and resistance to antimicrobial compounds, including antimicrobial peptides, NO, and oxidative killing. The potential live vaccine strains will be inoculated into mice by various routes (intradermal, intravenous, inhalation) and the immune response thoroughly characterized, including analysis of immune markers (cytokines, chemokines, immune cells) within infected tissue, and the antibody and T-cell responses. Our research plan involves a multidisciplinary approach and utilizes multiple investigators at several institutions in the region. This approach will allow us to propel the knowledge of F. tularensis pathogenesis and host response to a new level and identify novel vaccine candidates for the prevention of tularemia.